Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development.

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.

Inactivation of HIV-1 by chemical disinfectants: sodium hypochlorite.

The efficacy of sodium hypochlorite was assessed against human immunodeficiency virus type 1 suspended in low (8% v/v) or high (80% v/v) concentrations of serum or in a high (80%) concentration of blood. In the presence of 8% serum, 100 p.p.m. available chlorine in the disinfectant test mixture inactivated 3.75 log TCID50 HIV/ml within 30 s. When the test mixture contained 80% serum, 500 p.p.m. available chlorine inactivated more than 4 log TCID50 HIV/ml in 1-2 min. Lower concentrations of available chlorine were unable to inactivate the virus completely. In the presence of 80% blood, 1000 p.p.m. available chlorine in the disinfectant test mixture was unable to inactivate 3.75 log TCID50 HIV/ml, although 2500 p.p.m. available chlorine was able to inactivate at least 1.5 log TCID50 HIV/ml. In all test mixtures, the chlorine rapidly became combined and thus less active. Our results emphasise the importance of cleaning prior to disinfection with sodium hypochlorite since it may prove to be ineffective in the presence of high levels of organic matter. In cases where prior cleaning is impossible, care must be taken to use the higher recommended concentration (a minimum of 10,000 p.p.m. available chlorine).

Methodology for HIV disinfectant testing.

Due to the variation in protocols from studies by different workers for the inactivation of HIV by chemical disinfectants, only limited comparisons of the results can be made. These variations include those which apply to disinfectant testing in general, such as the level of organic load and the form of neutralization of the disinfectant, and those which apply particularly to HIV inactivation, such as the method used to detect infectious virus. Our suspension and carrier tests to assess the efficacy of chemical disinfectants against HIV are described and problems with the interpretation and applicability of the results are discussed.

Inactivation of human immunodeficiency virus type 1 by alcohols.

Alcohols are commonly used as disinfectants for skin, surfaces and immersion of some medical instruments. Measurements of the activity of alcohols against human immunodeficiency virus type 1 (HIV-1) must take account of the compatibility of neutralizers used to stop the disinfectant reaction, and of toxicity to the cell line used to detect residual virus. We have developed protocols to measure the efficacy of alcohols against HIV in suspension and dried onto surfaces in the presence of high and low protein concentrations. High titres of HIV in suspension were rapidly inactivated by 70% ethanol, independent of the protein load. When virus was dried onto a glass surface, the rate of inactivation decreased when high levels of protein were present. Due to its rapid evaporation, a spray or a wipe with alcohol cannot be guaranteed to disinfect a surface contaminated with blood or other body fluids without preliminary cleaning.

Survival of human immunodeficiency virus in suspension and dried onto surfaces.

Cell-free and cell-associated human immunodeficiency virus cultures suspended in 10% serum remained infectious for several weeks at room temperature. The stability was further increased when cell-associated virus was suspended in neat serum. When dried onto a glass coverslip, virus remained infectious for several days, although cell-associated virus lost infectivity more rapidly than cell-free virus.

Reversal of drug resistance in Trypanosoma cruzi and Leishmania donovani by verapamil.

Following previous studies of verapamil reversal of chloroquine resistance in malaria and multi-drug resistance in cancer cells, the effect of verapamil was investigated on nifurtimox-resistant Trypanosoma cruzi in vitro and antimony-resistant Leishmania donovani in vitro and in vivo. Verapamil alone was not active against either parasite, but in combination with nifurtimox it reversed the drug resistance of T. cruzi and in combination with sodium stibogluconate reversed the drug resistance of L. donovani.

The activity of nitrofurazone and furazolidone against Leishmania donovani, L. major and L. enriettii in vitro and in vivo.

Furazolidone and nitrofurazone showed in vitro activity against amastigotes of Leishmania donovani, L. enriettii and L. major in macrophages, at concentrations which were also toxic to the macrophages. A low grade of activity was observed against L. donovani infections in BALB/c mice by furazolidone but not with nitrofurazone. Nitrofurazone, in two concentrations, was not active when applied to the lesions of cutaneous leishmaniasis due to L. enriettii (guinea-pig infection) or L. major strain P (BALB/c mouse infection). After systemic administration to BALB/c mice infected with L. major strain JISH 252 clone 1, low-grade activity was observed at the highest level tested.

The influence of plasma concentrations of tri-iodothyronine on the acute increases in insulin and muscle protein synthesis in the refed fasted rat.

To examine whether the low plasma levels of triiodothyronine (T3) in fasted rats might limit the recovery of muscle protein synthesis on refeeding, rats were fasted for either 3 or 4 days and refed with or without pretreatment with thyroid hormones. Fasting suppressed T3 levels, plasma insulin and the rate of the translational phase of muscle protein synthesis (KRNA; the rate per unit RNA), especially after the 4-day fast. On refeeding, plasma T3 levels remained low for more than 3 h after the 3-day fast and for more than 8 h after the 4-day fast. Insulin concentrations increased within the first hour of refeeding, eventually achieving supranormal concentrations after the 3-day fast. The KRNA increased within the first hour of refeeding, achieving well-fed control values by 3 h after the 3-day fast or 24 h after the 4-day fast. The increases in KRNA were significantly correlated with the increases in insulin at low insulin concentrations, achieving a plateau value at 150 pmol/l, so that further increases in insulin were not associated with any further increases in protein synthesis. Pretreatment with thyroid hormone induced increased T3 levels which were maintained for up to 8 h of refeeding. This had no effect on the responses of either insulin or protein synthesis to refeeding after the 3-day fast, but did result in an acceleration of the recovery in the KRNA and plasma insulin levels in the rats fasted for 4 days. Analysis of the insulin-KRNA relationship showed no evidence for any increase in the insulin sensitivity of muscle protein synthesis with thyroid pretreatment, the initial stimulation of protein synthesis on refeeding the rats fasted for 4 days reflecting increased insulin secretion. Since in the untreated animals, insulin secretion on refeeding was also correlated with T3 levels, these results are consistent with the previously reported thyroidal dependence of insulin secretion.

Comparative studies of drug susceptibility of five strains of Trypanosoma cruzi in vivo and in vitro.

A comparative study showed that 5 laboratory strains of Trypanosoma cruzi could be divided into a non-responsive group (Sonya clone and Colombiana) and a responsive group (Tulahuén, Y and Peru), based on long-term treatment of mouse infections with nifurtimox and benznidazole. In vitro sensitivity of epimastigotes and blood-stream trypomastigotes in macrophage cultures did not distinguish the strains, nor did the rate of development of nifurtimox resistance by epimastigote cultures. 7 novel anti-T. cruzi compounds also behaved similarly with respect to the 2 groups. A small decrease in sensitivity was observed in vitro by non-responsive strains of T. cruzi after re-isolation from treated mice. It is postulated that there could be an immunological component involved in successful treatment of T. cruzi infection.

The effect of tri-iodothyronine administration on protein synthesis in the diabetic rat.

In young male rats diabetes caused decreases in circulating free tri-iodothyronine and RNA concentration in liver and muscle, and in the rate of protein synthesis per unit of RNA (RNA activity) in muscle. Tri-iodothyronine treatment significantly increased RNA concentrations, but not RNA activity, in these tissues. Thus: (1) impaired thyroid status is a component of the diabetic condition; (2) tri-iodothyronine cannot stimulate the translational phase of protein synthesis in the diabetic rat.

Vergelijking van de invloed van langdurige verstrekking van melk en lactose-vrije melk op de voedingstoestand van Bosnegerkinderen in Suriname in relatie tot het lactose-intolerantie-"probleem" / Comparison of the influence of prolonged supplying of milk and lactose-free milk on the nutritional status of Bushnegro children in Suriname in relation to the lactose-intolerance-"problem"

Report of a study concerning the practical meaning of hypolactasia (lactose-intolerance) in Surinamese Bushnegro children. At 2 boarding schools in the Surinamese interior, during the school year 1974 to 1975, each child was supplied daily with ca. 200 ml of milk. At one boarding school this was normal skimed milk and at the other lactose-free skimmed milk. The nutritional status was measured, both in the beginning and at the end of the school year, but no significant differences were found. Lactose-free milk had no better effect than the normal milk. There was also no improvement in the amount of hypolactasia. Thus, racial differences in the prevalence of hypolactasia seemed to have a genetic basis. Therefore, it is not caused by an adaptation to milk consumption. Combined results of weight tests proved that lactose-intolerance and milk-intolerance are not the same. It is concluded that normal, healthy Bushnegro children do tolerate and utilize milk in normal quantities